DiseaseWorldwide

AnnualDeaths

U.S  

Annual Cases Annual Deaths

 

All Infectious Diseases 17,000,000(1995)

n/a 165,750(1992)2

 

HIV/AIDS 2,300,000(1997)

71,293(1995)

32,655(1996)

 

Acute respiratory diseases 4,400,000(1995)

90,400,000(1994)

607(1996)

 

Foodbourne illnesses 3,100,000(1995)

6,000,000(1983)

9,000(1983)

 

Malaria 2,000,000(1995)

n/a n/a  

Tuberculosis 3,100,000(1995)

22,860(1995)

1,194(1996)

 

Hepatitis B 1,100,000(1995)

10,805(1995)

3,811(1996)

 

Chlamydia n/a 4,000,000(1996)3

n/a  

1 Sources include the World Health Organization and the Centers for Disease Control and Prevention (CDC).

 

Cases and Mortality Rates for Infectious Diseases

Ghost E. coli

Inflammation,

Schock

Immune stimulation- adjuvans- activity

Carrier-Targeting

Major immunological questions:

Endotoxic activities, therapeutic window, routes of administration

Routes of activation comparing LPS, uptake by APCs

Stimulation of immune responses

Possibility for packaging, targeting to specific cells

Bacterial Cell Envelopes, LPS, Lipid A partial- structures

Avanti's New Potent Vaccine Adjuvant

Natural (Salmonella Minnesota, R595

 monophosphoryl derivative

endotoxically inactive

acting as an adjuvant

inducing tolerance to Salmonella enteritidis LPS and tumor necrosis factor alpha (TNF-alpha)

Detoxified Lipid A

Structure LPS

1,E+00 1,E+02 1,E+04 1,E+06 1,E+08

EU/mg

S-layer ( B.sphaericus)

S-layer ( B. stear.)

Ghosts (E.coli O26:B6)

Ghosts (S.typhim.)

LPS (E.coli O26:B6)

LPS ( S. ab. equi )

Control

Limulus Test, Endotoxicity

0

2.000

4.000

6.000

8.000

10.000

0,00001 0,0001 0,001 0,01 0,1 1 10 100

[g/ml]

TN

F [

pg

/ml]

LPS

MDPghost

S-layer(B.sph) S-layer(B.st.)

Stimulation of TNFa by bacterial ghostsin Mo

Bacterial ghosts prepared from Escherichia coli O26:B6 and Salmonella typhimurium C5 induce dose-dependent antibody responses against bacterial cells or their corresponding lipopolysaccharides (LPS) in doses 25 ng kg-1 when administered intravenously to rabbits. No significant fever responses in rabbits have been recorded in doses of < 250 ng kg-1 E. coli O26:B6 ghosts and up to doses of 250 ng kg-1 S. typhimurium C5 ghosts when applying test methods recommended by the US pharmacopoeia.

Vaccine 1997 Feb;15(2):195-202

Endotoxicity does not limit the use of bacterial ghosts as candidate vaccines.

Hypothesis:

transmembrane integrin glycoproteins e.g.Mac-1 serve as a signaling partner for glycolipid-linked glycoproteins that lack trans-membrane and cytoplasmic domains.

LPS, CD14

0 5 10 15 20 25

TNF[ng/ml] LPS FCS aCD14 p< (ng/ml) (ug/ml)

10 + - 10 - -

10 + 0,5 0,05 10 - 0,5

10 + 0,1 0,02 10 - 0,1

100 + - 100 - -

100 + 0,5 0,02 100 - 0,5 0,02

100 + 0,1 0,05 100 - 0,1 0,05

- +/- -

- +/- +/-

Inhibition of TNFa by anti CD14

CD14GPIToll-

likeR-4

LPS- LBP

Stat3,1,4

HSP60?

NFkB

IL-6, IFNy,...

MAPK/ p38 JNK

ERK MAPK

CR3

MAC1

?

R

Janus

Kinase

CREB,

ATF1

?

PLC?

...,myk,Elk, Mnk

eIF4E AH,10/2000

P

Pathways for LPS stimulation

Uptake of ghosts in RAW-Mo

GFP labelled Ghost, RAW cells

FISH, GFP mRNA in RAW Mo

0

20

40

60

80

100

120

Co Ghosts5; 0,5 ul Ghost/SB Ghosts/G5

% o

f Con

trol

LPS: Co, 0,001; 0,01; 0,1 ug/ml , Co:50-200 pg/ml TNFa, 100%: 5-20 ng/ml TNFa

Ghosts: E. Coli, 5, 0,5 ul; 100 %: 5-20 ng/ml TNFa

SB203580: 5, 10, 20 uM; G5: Protein kinase CK2 inhibitor, 5,20, 50 uM

0

20

40

60

80

100

120

140

1 2 3 4 5 6 7

% of

Co

Inhibition of LPS stimulated TNFa Inhibition of Ghost stimulated TNFa

by kinase inhibitors in supernatant by kinase inhibitors packed inside ghosts

LPS

SB/G5

APCs and T-cells

  mRNA Isolation: Quick PrepMicro mRNA Purification Kit mRNA semiquantification:Nuleic dotMetricTM Basic Kit (Geno Technology) 

Reverse transcripton reaction RT- PCR:LightCyclerTM System (Roche), FastStart DNA Master SYBR Green I (Roche) Quantification:external cDNA standards with known amounts of initial copy number over a 105 - fold range were coamplified calculation with LightCycler Quantification Software v3.39 during the log- linear phase of the amplification  ß- actin was used to normalize for inefficiencies in cDNA synthesis  

Quantitative Real Time PCR( Light Cycler System )

-4

24

0

4

8

12

16

20

71 73 75 77 79 81 83 85 87 89 91 93

no template

-dF/

dT

Temperature (°C)

B

32

-202468

1012141618202224262830

400 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38

106 105

104 103

102

no template

Cycle number

Fluo

resc

ence

A

Amplification in RT- PCR

IL-12

TNFa

Eif2-by

M Co 3 6 16 16 hrs

IL-12, TNFa mRNA in Ghost- stimulated THP-1 MO (RT- PCR)

1

10

100

1000

0h 4h 16h

pg/m

l

TNFa

IL-12LPS

Ghosts o

Stimulation IL-12 and TNFa in THP1 cellsELISA

10

100

1000

Co 0,001 0,01 0,1 1 10

ug/ml

pg/m

l

TNFa

IL-12

Stimulation of IL-12 and TNFa in PBM

Immune mediators in DC derived from PBM ( IL4, GMCSF, 5-7 d )

secrete large amounts of inflammatory cytokines as precursors

high Ag capture capacity at their immature stage

stimulation of quiescient, B and T lymphocytes

low levels of antigen to induce strong T cell response

Adherence of ALEXA labeled ghosts

0

200

400

600

800

1000

1200

1400

Control LPS Ghosts

TN

F-

(p

g/m

l)

0

200

400

600

800

1000

Control LPS Ghosts

TNF-

(pg/

ml)

Stimulation of TNFa in PBM and DC

0

50

100

150

200

250

300

350

Control LPS Ghosts

IL-1

2 p7

0+p4

0 (p

g/m

l)

0

5000

10000

15000

20000

25000

Control LPS Ghosts

IL-1

2 p7

0+p4

0 (p

g/m

l)

Stimulation of IL-12 ( p40, p70) protein in PBM and DC

0

10

20

30

40

50

60

Control LPS Ghosts

IL-1

2 p

40 r

ela

tive

mR

NA

le

ve

l

Stimulation of IL-12 p40 mRNA in DC

0

10

20

30

40

50

Control LPS Ghosts

IL-1

8 r

ela

tiv

e m

RN

A l

ev

el

Stimulation of IL-18 relative mRNA in DC

What are the mechanisms for the good activation of IL-12 in APC, especially DC ?

Does this have a clinical value ?

Ghosts and DC

Microbial stimuli that promote DC maturation

Bacteria

( E. coli, M. tuberculosis, Staph. aureus, Strept. ,...

Protozoa

LPS

Bacterial DNA,

Viral and doubled-stranded RNA

Bacterial heat shock proteins

Migration

Exit of activated DCs from peripheral sites

Entry into the T cell areas of secondary lymphoid tissues

Antigen presentation

Upregulation of antigen presenting molecules (MHC class I and class II, CD1)

Delivery of antigen to the MHC class I pathway

Upregulation of molecules involved in interaction with T lymphocytes

Costimulatory molecules (B7-1, B7-2)

Adhesion molecules (ICAM-1, VLA-4)

Signalling molecules (CD40)

Production of cytokines

Induction of IL-12, TNF, IL-10, IL-6, IFN-/

Recruitment of DC precursors to peripheral sites

Increased transient survival of DCs in the absence of growth signals

Irreversible maturation followed by apoptotic death

Activation of DC functions by microbial stimuli.

0,75

1,25

1,75

2,25

1:10 1:30 1:60 1:100 1:300 1:600 1:1000

Ratio DC : T cell

ab

sorb

an

ce (

A45

0-A

690)

iDCs mDCs

mDCs+G GBH BF

Pavol KUDELA

maturation mix (MM)-TNF-; IL-1; PGE2; IL-6; GM-CSF; IL-4

MM + bacterial ghosts + GM-CSF + IL-4

Mature DCs and ghosts induce strong T-cell responses, MLR

MHC class I and II, CD11a,b,c, CD50,

CD54, CD58

Activation of T- cells

IL-12, T cell attractant chemokine

Induce ghosts differentiation and activation of DC functions ?

Gut 2000 Jul;47(1):79-87

Non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial cell/leucocyte co-cultures.

Haller D, Bode C, Hammes WP, Pfeifer AM, Schiffrin EJ, Blum S.

Institute of Biological Chemistry and Nutrition Science, University Hohenheim, Germany.

BACKGROUND AND AIM: Intestinal epithelial cells (IEC) are thought to participate in the mucosal defence against bacteria and in the regulation of mucosal tissue homeostasis

Challenge of CaCO-2 cells with non-pathogenic E coli and Lactobacillus sakei induced expression of IL-8, MCP-1, IL-1beta, and TNF-alpha mRNA in the presence of underlying leucocytes.

Leucocyte sensitised CaCO-2 cells produced TNF-alpha and IL-1beta whereas IL-10 was exclusively secreted by human peripheral blood mononuclear cells.

CaCO-2 cells alone remained hyporesponsive to the bacterial challenge.

CONCLUSION: The differential recognition of non-pathogenic bacteria by CaCO-2 cells required the presence of underlying leucocytes. These results strengthen the hypothesis that bacterial signalling at the mucosal surface is dependent on a network of cellular interactions.

Microbiol Immunol 1999;43(10):925-35

Cytokine secretion by stimulated monocytes depends on the growth phase and heat treatment of bacteria: a comparative study between lactic acid bacteria and invasive pathogens.

Haller D, Bode C, Hammes WP.

Hohenheim University, Stuttgart, Germany. dirk.haller@rdls.nestle.com

The challenge of monocytes with three LAB strains, Listeria monocytogenes or enterohaemorrhagic Escherichia coli (EHEC) elicited a strain specific, dose-dependent biphasic TNF-alpha secretion.

LPS exhibited a higher capacity to stimulate monocytes than purified gram positive cell walls or muramyldipeptide.

 

In comparison to pathogenic bacteria, the maximal secretory TNF-alpha response (TNFmax) was up to 2 fold higher with LAB strains.

 

In general, the amount of bacteria (EDmax) necessary to induce maximal TNF-alpha secretion (TNFmax) was approximately 1 to 3 log higher for heat killed bacteria when compared to live bacterial cells illustrating the significant lower potential of heat killed bacteria to activate monocytes.

Bacterial cell envelopes

for vaccine development

• No endotoxic limitations

• lipid A activation pathways, CD14, p38 MAP kinase

• provide adjuvans activity comparable to lipid A structures

• effective uptake by phagocytes ( others by different mechanisms ?)

• potent inducers of IL-12, ( IL-18 )

• may propagate maturation of dendritic cells

• may enable packaging of systemic toxic compounds and targeting

to specific cells or organsAH, 10/2000

M. Szostak

Horst Mader

M. Schmalnauer

Gudrun Kohl

Fürst Ladani

Beate Mayr

Margit Weghofer